This invention relates to a method of enzyme-mediated breakdown of fibrinogen and fibrin. More particularly, the invention relates to a method for degrading fibrinogen and causing lysis of fibrin clots through mediation by a fibrinolytic matrix metalloproteinase. The invention further relates to the use of a fibrinolytic matrix metalloproteinase as an antithrombotic to reconstruct stenotic vessels and remove fibrin deposits.
The clotting of blood is part of the body's natural response to injury or trauma. Blood clot formation derives from a series of events called the coagulation cascade, in which the final steps involve the formation of the enzyme thrombin. Thrombin converts circulating fibrinogen into fibrin, a mesh-like structure that forms the insoluble framework of the blood clot. As a part of hemostasis, clot formation is often a life-saving process in response to trauma and serves to arrest the flow of blood from severed vasculature.
The life-saving process of clot production in response to an injury can become life-threatening when it occurs at inappropriate places in the body. For example, a clot can obstruct a blood vessel and stop the supply of blood to an organ or other body part. In addition, the deposition of fibrin contributes to partial or complete stenosis of blood vessels, resulting in chronic diminution of blood flow. Equally life-threatening are clots that become detached from their original sites and flow through the circulatory system causing blockages at remote sites. Such clots are known as embolisms. Indeed, pathologies of blood coagulation, such as heart attacks, strokes, and the like, have been estimated to account for approximately fifty percent of all hospital deaths.
The formation of fibrin during inflammation, tissue repair, or hemostasis, plays only a temporary role and must be removed when normal tissue structure and function is restored. Thus, a fibrin clot that forms quickly to stop hemorrhage in an injured blood vessel is remodeled and then removed to restore normal blood flow as healing occurs. The system responsible for fibrin breakdown and clot removal is the fibrinolytic system. Action of the fibrinolytic system is tightly coordinated through the interaction of activators, zymogens, enzymes, as well as through inhibitors of each of these components, to provide focused local activation at sites of fibrin deposition (Francis et al. 1994; Collen 1980; Collen et al. 1991).
The principal mediator of fibrinolysis is plasmin, a trypsin-like endopeptidase that cleaves fibrin to dissolve clots and to permit injured tissues to regenerate. Plasmin has also been demonstrated to play a role in degrading proteins involved in cell--cell and cell-matrix interactions, as well as in activating other tissue remodeling enzymes such as matrix metalloproteinases (Murphy et al. 1992). In turn, control of plasmin activity, as well as these other extracellular events, is principally mediated by plasminogen activators, which convert the inactive zymogen plasminogen to the active enzyme plasmin.
In clinical settings it is commonly desirable to activate or potentiate the fibrinolytic system. This is particularly necessary in cases of myocardial infarction in which coronary arteries become occluded and require recanalization. Catheterization has proven somewhat effective in such recanalization, but pharmacologic agents are desired to supplement or replace such invasive procedures to inhibit reocclusion. The study of the intricate system of thrombolysis and fibrinolysis has been a rapidly growing field, which has resulted in the development of a new generation of thrombolytic agents.
Previous therapeutic treatments for dissolving life-threatening clots have included injecting into the blood system various enzymes that are known to break down fibrin (Collen 1996). The problems with these treatments has been that the enzymes were not site-specific, and, therefore, would do more than just cause dissolution of the clot. In addition, these enzymes interfere with and destroy many vital protein interactions that serve to keep the body from bleeding excessively due to the many minor injuries it receives on a daily basis. Destruction of these safeguards by such enzymes can lead to serious hemorrhage and other potentially fatal complications.
Currently, the best known therapeutic agents for inducing or enhancing thrombolysis are compounds that cause the activation of plasminogen, the so-called "plasminogen activators" (Brakman et al. 1992). These compounds cause the hydrolysis of the arg560-val561 peptide bond in plasminogen. This hydrolysis yields the active two-chain serine protease, plasmin. A number of such plasminogen activators are known, including serine proteases such as urokinase plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), streptokinase (a non-enzyme protein) and staphylokinase. Of these, streptokinase is the most widely used therapeutic thrombolytic agent. However, while streptokinase and the other plasminogen activators have proven helpful in recanalization of coronary arteries, their ability to improve mortality is not devoid of side effects and their use still requires stringent control conditions to achieve success in a high percentage of cases (Martin et al. 1994). In addition, the use of such compounds can cause bleeding complications in susceptible individuals. On the other hand, one of the drawbacks of the use of t-PA in clinical trials has been the early reformation of the clot after it has been dissolved, resulting in thrombotic reocclusion in some patients.
Numerous studies have documented the ability of t-PA to initiate or potentiate thrombolytic phenomena (Sobel et al. 1987). As a result, t-PA, specifically its recombinant form, rt-PA, is becoming more popular as a thrombolytic pharmaceutical. Nonetheless, rt-PA does suffer from serious limitations, including extremely high dosage cost, and variable efficacy. In addition, specific rapid-acting inhibitors of t-PA have been identified in human plasma and other fluids (Collen et al. 1987). A further approach to t-PA involves the potential use of gene transfer of and expression of recombinant t-PA in endothelial cells (Lee et al. 1993). This procedure is exceedingly complex and is not likely to be practical as a thrombolytic treatment in the near future.
Enzymes other than plasmin are also known that can degrade fibrin(ogen) to different extents. For example, endogenous leukocyte proteases (Bilezikian et al. 1977; Plow et al. 1975), later identified as elastase and cathepsin-G (Gramse et al. 1978; Plow 1980; Plow et al. 1982), can partially degrade fibrin(ogen). Other enzymes are also known that degrade fibrin. Such enzymes include hemolytic enzymes collected from the venom of certain snakes, e.g., the families crotalidae and viperidae (Purves et al. 1987; Retzios et al. 1992; Sanchez et al. 1991). Fibrinolytic enzymes isolated from snakes can be grouped into two different classes (Guan et al. 1991). Those enzymes that preferentially degrade the A.alpha.-chain of fibrinogen and also the .alpha.- and .beta.-chains of fibrin are zinc metalloproteases (Guan et al. 1991) and all can be inhibited by EDTA. Enzymes in the second class are serine proteinases, and exhibit specificity for the .beta.-chain of fibrin (Guan et al. 1991). An endopeptidase from puff adder venom (Bitis arietans) can cleave at the .gamma.-chain cross-linking site and thereby cleave Fragment D-dimer into a D-like monomer (Purves et al. 1987). Fibrinolytic enzymes have also been obtained from leeches (Zavalova et al. 1993; Budzynski 1991), as well as from the growth medium of a bacterium (Aeromonas hydrophila) that was recovered from leech intestinal tract (Loewy et al. 1993).
The matrix metalloproteinases ("MMPs" or "matrixins") are a class of enzymes that are expressed within the connective tissues of vertebrates. The MMPs occur natively in such tissues and play critical roles in the continuous processes associated with the laying down and remodeling of the extracellular matrix (ECM), hence their name: "matrix" metalloproteinases. The MMPs can be characterized as "intrinsic" or "endogenous" enzymes insofar as their proper function is within the tissues of the organism in which they are natively expressed. As such the MMPs are distinguished functionally and evolutionarily from the metalloproteinases found in snake venom and the like, which function outside the organism in which they are expressed, and may therefore be designated "exogenous" enzymes.
The matrix metalloproteinases are presently divided into six classes of enzymes: collagenases, gelatinases, stromelysins, matrilysin (formerly PUMP), metalloelastase, and membrane-type metalloproteinase. The collagenase class of MMPs includes interstitial collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase-3 (MMP-13). The gelatinase class of MMPs includes gelatinase-A (MMP-2) and gelatinase-B (MMP-9). The stromelysin class of MMPs includes stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11). The matrilysin class of MMPs includes matrilysin (MMP-7). The metalloelastase class of MMPs includes metalloelastase (MMP-12). The membrane-type metalloproteinase class of MMP includes membrane-type metalloproteinase (MMP-14). For a recent review see Nagase (1996).
MMPs are known to proteolytically cleave and degrade a number of proteins and proteoglycans that are associated with the extracellular matrix (ECM) of connective tissue. They have been shown to break down a number of proteins including collagen (Types I-IV, VII and X), laminin, fibronectin, elastin and proteoglycans. MMPs have also been identified in leukocytes (Welgus et al. 1990). It has been shown that MMP-2 and MMP-9 possess elastase activity (Senior et al. 1991), to which some of the complex proteolytic activity, initially observed in granulocytes, could be attributed (Sterrenberg et al. 1983). In addition, MMP-12 has also been shown to possess elastin activity (Nagase 1996). MMP-7 has been shown to possess strong proteolytic activity and digests aggrecan, cartilage link protein, fibronectin, and elastin (Nagase 1996). MMPs participate in the remodeling of tissues in physiological processes such as morphogenesis and embryonic development, as well as in the pathophysiology of wound healing, tumor invasion, and arthritis (Matrisian 1992; Nagase et al. 1991; Woessner 1991; Werb et al. 1992).
The expression of MMPs and their inhibitors is under extensive and varied molecular and cellular control (Kleiner et al. 1993; Matrisian 1992; Woessner 1991). Known regulating factors include hormones, cytokines, proto-oncogenes, steroids, and growth factors. MMPs are blocked by specific inhibitors called "tissue inhibitors of metalloproteinases" (TIMPs) that can block the activity of each member of the family. An enzyme inhibitor complex is formed and no turnover of connective tissue takes place if the MMPs are present in excess. The main focus of research on ECM has been to limit ECM degradation by MMPs to interrupt or interfere with the progression of disease states. Several groups of investigators are making small molecules that could inhibit proteinases to alter their destructive activity in arthritis, and as antiangiogenic factors to inhibit tumor spread.
Matrix metalloproteinase 3 (MMP-3) belongs to the stromelysin class of matrix metalloproteinases. MMP-3 is expressed in mature macrophages (Campbell et al. 1991), but also in endothelial cells, smooth muscle cells and fibroblasts. More recently, MMP-3 has been shown to be expressed in macrophage-derived foam cells from experimental atheroma (Galis et al. 1995). The inactive zymogen, proMMP-3, is activated by neutrophil elastase, plasma kallikrein, plasmin, chymotrypsin, trypsin, cathepsin G, and mast cell tryptase, as well as by mercurial compounds, such as 4-aminophenylmercuric acetate (APMA) (Nagase et al. 1992; Kleiner et al. 1993; Nagase et al. 1990; Nagase 1991). Elevated levels of MMP-3 have been found in the joints of patients suffering from osteoarthritis and rheumatoid arthritis. In atherosclerotic plaques there is a large amount of fibrin(ogen)-related antigen (FRA) consisting of different molecular forms (Bini et al. 1987; Bini et al. 1989; Smith et al. 1990; Valenzuela et al. 1992). Two very recent studies have shown the presence of matrix metalloproteinase 3 in atherosclerotic plaques (Henney et al. 1991; Galis et al. 1994), but its function in this context has remained unelucidated. Indeed, MMP-3 has been viewed in these studies as a potential negative factor.
The known substrates of MMP-3 include proteoglycans, collagen type IV, fibronectin, and laminin. Such substrates are typical of matrix metalloproteinases in general (Doolittle 1987). There has been no suggestion, however, that any matrix metalloproteinases might be involved in the degradation of fibrinogen or fibrin. Nor has there been any indication that matrix metalloproteinases could be used for fibrinolysis or thrombolysis.
From the foregoing discussion, it becomes clear that significant gaps exist in the understanding of processes involved in thrombus formation and degradation. While certain approaches have been identified that permit a measure of control over these processes, these approaches suffer serious deficiencies related to cost, efficacy, or safety. The diagnosis and treatment of disease states associated with physiological processes involving fibrinogen and fibrin have also been found lacking.
As a result, there exists a need for effective compositions and methods for use in limiting thrombus development and inducing thrombolysis.
There is a need for methods of disrupting blood clots and atherosclerotic plaques, both in vitro, such as for diagnostic purposes, and in vivo, such as for therapeutic treatment of embolism, atherosclerosis and other clinically important disorders.
In addition, there exists a need for diagnostic and experimental materials and methods for revealing more information concerning the physical and chemical processes involved in thrombus formation and degradation.
Moreover, there is a need for effective treatment to restore at least some integrity to a damaged vessel wall, to promote regression of atherosclerotic plaques, and to aid in angioplasty and bypass surgery to prevent reocclusion.